Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore. We used wild-type PYP and the mutant E46A, in protonated and deprotonated states (referred to as pE46A and dpE46A, respectively), to reduce the number of hydrogen bond interactions between the pCa phenolate oxygen and the protein and to vary the negative charge density in the chromophore-binding pocket. Their effects on the yield and rate of chromophore isomerization were determined by ultrafast spectroscopy. Molecular dynamics simulations were used to relate these results to structural changes in the mutant protein. We found that deprotonated pCa in E46A has a slower isomerization rate as the main part of this reaction was associated with time constants of 1 and 6 ps, significantly slower than the 0.6 ps time constant in wild-type PYP. The quantum yield of isomerization in dpE46A was estimated to be 30 ± 2%, and that of pE46A was 32 ± 3%, very close to the value determined for wtPYP of 32 ± 2%. Relaxation of the isomerized product state I0 to I1 was faster in dpE46A. We conclude that the negative charge on pCa stabilized by the short hydrogen bonds with E46 and Y42 affects the rate of isomerization but not the quantum yield of isomerization. With this information, we propose a scheme for the potential energy surfaces involved in the isomerization and suggest protein motions near the pCa backbone as key events in successful isomerization.